Search

Multidrug-resistant (MDR) Gram-negative bacterial infections have emerged as a major public health concern. The aim of the present study was to detect the rate of infections due to MDR Gram-negative bacteria (GNB) in a tertiary care hospital, the rate of Carbapenemases and AmpC-b-lactamases production and the Antimicrobial susceptibility test pattern (AST) among MDR GNB. The rate of MDR GNB during the study period was 25.70%. Urine samples showed the highest contribution to the total MDR GNB. Among the total MDR GNB isolates, 166 were randomly selected and included in the present study. A higher rate of MDR GNB was reported among male patients (61.5%) compared to the females (38.5%) and most of them were from the patients aged between 61-70 years (30.7%). The most prevalent MDR GNB was Klebsiella pneumoniae 80 (48.12%), followed by Escherichia coli 43 (25.9%). AST of MDR GNB revealed their significant resistance to b-lactamases/b-lactamases inhibitors, cephalosporins, fluoroquinolones and carbapenem drugs (98%). Of 123 MDR Enterobacterales, 83% of them were found to be Metallo b-lactamase (MBLs) producers by mCIM and eCIM methods. Of 43 MDR non-fermenters, 29 (67.4%) of them were found to be carbapenemase producers by MHT. About 29.51% of MDR GNB isolates were found to be AmpC producers by AmpC disk test. A reliable and rapid phenotypic method to detect carbapenemases and AmpC b-lactamases among MDR GNB in a routine microbiology laboratory method is clinically important to guide antibiotic therapy and implementation of effective infection control practices. [ABSTRACT FROM AUTHOR]

Objective To identify early Klebsiella pneumoniae (KP) infection after liver transplantation and its impact on prognosis. Methods Clinical data of 171 liver transplant recipients were retrospectively analyzed, and they were divided into the non-infection (n=52) and infection groups (n=119) according to the bacterial culture results at postoperative 2 weeks. In the infection group, KP was not detected in 86 cases (non-KP infection group), and KP was cultured in 33 cases (KP infection group). Preoperative, intraoperative and postoperative data were statistically compared between the non-infection and infection groups, and between the non-KP infection and KP infection groups. The risk factors of early KP infection after liver transplantation and the influencing factors of long-term survival of the recipients were analyzed.Results Compared with the non-infection group, model for end-stage liver disease (MELD) score and total bilirubin level were higher, the operation time was longer, the length of postoperative intensive care unit (ICU) stay and the length of hospital stay were longer, the amount of intraoperative red blood cell transfusion was higher, the hospitalization expense was higher, the incidence of severe complications was higher, white blood cell count, absolute neutrophil cell count and neutrophil-to-lymphocyte ratio at postoperative 14 and 30 d were higher, absolute lymphocyte count at postoperative 14 d was lower and hemoglobin level at postoperative 30 d was lower in the infection group. The differences were statistically significant (all P<0.05). Compared with the non-KP infection group, MELD score, total bilirubin level and aspartate aminotransferase (AST) level were higher, the operation time and the length of postoperative ICU stay were longer, the hospitalization expense was higher, the 90-d fatality was higher, the albumin level at postoperative 14 d was lower, and total bilirubin level at postoperative 30 d was higher in the KP infection group. The differences were statistically significant (all P<0.05). Among 33 recipients with KP infection, 16 cases were resistant to carbapenem antibiotics, and 7 of them died within postoperative 90 d. Seventeen cases were intermediate or sensitive to carbapenem antibiotics, and 4 of them died within postoperative 90 d. Preoperative MELD score ≥17 and operation time≥415 min were the independent risk factors for KP infection after liver transplantation (both P<0.05). The length of postoperative ICU stay ≥44 h and KP infection were the independent risk factors for long-term prognosis of liver transplantation (both P<0.05). Conclusions KP infection is an independent risk factor for death after liver transplantation. High preoperative MELD score and long operation time are the independent risk factors for early KP infection after liver transplantation. [ABSTRACT FROM AUTHOR]

Purpose: Carbapenem resistant Pseudomonas aeruginosa (CR-PA) is escalating worldwide and leaves clinicians few therapeutic options in recent years, β-lactam/β-lactamase inhibitor combinations (ceftolozane-tazobactam, ceftazidime-avibactam) and a new siderophore cephalosporin (cefiderocol) have been approved for the treatment of P. aeruginosa infection and have shown potent activity against isolates defined as carbapenem resistant. The aim of this study was to determine the phenotypic profile of these agents against CR-PA in the emerging setting of carbapenemases. Methods: CR-PA clinical isolates were collected from three teaching hospitals in different geographical regions between January 2017-December 2021. All isolates were subjected to phenotypic carbapenemase testing using modified carbapenem inactivation method. MICs were determined by reference broth microdilution and evaluated according to EUCAST standards, while genotypic profiling was determined using PCR methods. Results: 244 CR-PA sourced most frequently from the respiratory tract (32.2%), blood (20.4%) and urine (17.5%) were evaluated. Of all isolates, 32 (13.1%) were phenotypically and 38 (15.6%) were genotypically defined as carbapenemase-positive. The most common carbapenemase was GES (63.1%), followed by VIM (15.8%). The MIC50/90(S%) of ceftazidime/avibactam, ceftolozane/tazobactam and cefiderocol in all CR-PA isolates were 4 and 32 (80%), 1 and > 64 (69%) and 0.25 and 1 mg/L (96%), respectively. Cefiderocol was also the most active agent in carbapenemase-positive isolates (90%). Conslusion: While ceftolozane/tazobactam and ceftazidime/avibactam remained highly active against CR-PA devoid of carbapenemases, cefiderocol provided potent in vitro activity irrespective of carbapenemase production. When considering the potential clinical utility of newer agents against CR-PA, regional variations in carbapenemase prevalence must be considered. [ABSTRACT FROM AUTHOR]

Background: Klebsiella pneumoniae species complex (KpSC) is an important disseminator of carbapenemase‐encoding genes, mainly blaKPC‐2 and blaNDM‐1, from hospitals to the environment. Consequently, carbapenem‐resistant strains can be spread through the agrifood system, raising concerns about food safety. This study therefore aimed to isolate carbapenem‐resistant KpSC strains from the agricultural and environmental sectors and characterize them using phenotypic, molecular, and genomic analyses. Results: Klebsiella pneumoniae and Klebsiella quasipneumoniae strains isolated from soils used for lemon, guava, and fig cultivation, and from surface waters, displayed an extensive drug‐resistance profile and carried blaKPC‐2, blaNDM‐1, or both. In addition to carbapenemase‐encoding genes, KpSC strains harbor a broad resistome (antimicrobial resistance and metal tolerance) and present putative hypervirulence. Soil‐derived K. pneumoniae strains were assigned as high‐risk clones (ST11 and ST307) and harbored the blaKPC‐2 gene associated with Tn4401b and Tn3‐like elements on IncN‐pST15 and IncX5 plasmids. In surface waters, the coexistence of blaKPC‐2 and blaNDM‐1 genes was identified in K. pneumoniae ST6326, a new carbapenem‐resistant regional Brazilian clone. In this case, blaKPC‐2 with Tn4401a isoform and blaNDM‐1 associated with a Tn125‐like transposon were located on different plasmids. Klebsiella quasipneumoniae ST526 also presented the blaNDM‐1 gene associated with a Tn3000 transposon on an IncX3 plasmid. Conclusion: These findings provide a warning regarding the transmission of carbapenemase‐positive KpSC across the agricultural and environmental sectors, raising critical food safety and environmental issues. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The healthcare water environment is a potential reservoir of carbapenem-resistant organisms (CROs). To report the role of the water environment as a reservoir and the infection control measures applied to suppress a prolonged outbreak of Klebsiella pneumoniae carbapenemase-producing Serratia marcescens (KPC-SM) in two intensive care units (ICUs). The outbreak occurred in the ICUs of a tertiary hospital from October 2020 to July 2021. Comprehensive patient contact tracing and environmental assessments were conducted, and a case–control study was performed to identify factors associated with the acquisition of KPC-SM. Associations among isolates were assessed via pulsed-field gel electrophoresis (PFGE). Antibiotic usage was analysed. The outbreak consisted of two waves involving a total of 30 patients with KPC-SM. Multiple environmental cultures identified KPC-SM in a sink, a dirty utility room, and a communal bathroom shared by the ICUs, together with the waste bucket of a continuous renal replacement therapy (CRRT) system. The genetic similarity of the KPC-SM isolates from patients and the environment was confirmed by PFGE. A retrospective review of 30 cases identified that the use of CRRT and antibiotics was associated with acquisition of KPC-SM (P < 0.05). There was a continuous increase in the use of carbapenems; notably, the use of colistin has increased since 2019. Our study demonstrates that CRRT systems, along with other hospital water environments, are significant potential sources of resistant micro-organisms, underscoring the necessity of enhancing infection control practices in these areas. [ABSTRACT FROM AUTHOR]

Background: Carbapenem resistance in Pseudomonas aeruginosa is a serious global health problem. We investigated the clonal distribution and its association with the carbapenem resistance mechanisms of carbapenem-non-susceptible P. aeruginosa isolates from three Korean hospitals. Methods: A total of 155 carbapenem-non-susceptible P. aeruginosa isolates collected between 2011 and 2019 were analyzed for sequence types (STs), antimicrobial susceptibility, and carbapenem resistance mechanisms, including carbapenemase production, the presence of resistance genes, OprD mutations, and the hyperproduction of AmpC ß-lactamase. Results: Sixty STs were identified in carbapenem-non-susceptible P. aeruginosa isolates. Two high-risk clones, ST235 (N=41) and ST111 (N=20), were predominant; however, sporadic STs were more prevalent than high-risk clones. The resistance rate to amikacin was the lowest (49.7%), whereas that to piperacillin was the highest (92.3%). Of the 155 carbapenem-non-susceptible isolates, 43 (27.7%) produced carbapenemases. Three metallo-β-lactamase (MBL) genes, blaIMP-6 (N=38), blaVIM-2(N=3), and blaNDM-1(N=2), were detected. blaIMP-6 was detected in clonal complex 235 isolates. Two ST773 isolates carried blaNDM-1 and rmtB. Frameshift mutations in oprD were identified in all isolates tested, regardless of the presence of MBL genes. Hyperproduction of AmpC was detected in MBL gene-negative isolates. Conclusions: Frameshift mutations in oprD combined with MBL production or hyperproduction of AmpC are responsible for carbapenem resistance in P. aeruginosa. Further attention is required to curb the emergence and spread of new carbapenem-resistant P. aeruginosa clones. [ABSTRACT FROM AUTHOR]

Background: The spread of Carbapenemase-producing Organisms (CPO) remains a major threat globally. Within clinical settings, the existing method of determining gene load involves traditional culture to determine bacterial load and polymerase-chain-reaction-based Xpert Carba-R Assay to determine carbapenemase gene type. However, there is a need for a fast and accurate method of quantifying CPO colonisation to study the risk of persistent CPO carriage. Objective: This study evaluated the accuracy of Xpert Carba-R Ct value in estimating carbapenamase producing bacterial loads in stool samples. Methods: Stool samples were obtained from an ongoing study investigating the household transmission of CPO in Singapore. Stool samples lacking carbapenemase producing organisms were spiked with organism carrying a single carbapenemase gene (blaKPC, blaNDM, blaVIM, blaOXA-48(-like) or blaIMP-1) and serially diluted before being subjected to Xpert Carba-R assay and traditional culture. Standard curves with regression lines showing correlation between Ct values and plate counts were generated. The standard curves were validated with stool samples collected from patients. Results: The limit of detection of blaNDM, blaKPC, and blaOXA-48 was approximately 103 cfu/mL, while that of blaIMP-1 and blaVIM was approximately 104 cfu/mL. Validation of the blaNDM and blaOXA-48 curves revealed average delta values of 0.56 log(cfu/mL) (95% CI 0.24–0.88) and 0.80 log(cfu/mL) (95% CI 0.53–1.07), respectively. Conclusions: Our validation data for stool positive for blaNDM and blaOXA-48-type suggests that bacterial loads can be estimated within a reasonable range of error. [ABSTRACT FROM AUTHOR]

Introduction: Enterobacter chengduensis was defined as a novel species in the genus. Enterobacter in 2019, however, antimicrobial resistance, such as carbapenem resistance, has rarely been described in E. chengduensis. This study described the molecular features of four carbapenem-resistant E. chengduensis strains collected from a tertiary health care hospital in Southwest China. Methods: Whole genome sequencing (WGS) was used to determine the genome sequence of four E. chengduensis strains. The precise species of strains were identified by average nucleotide identity (ANI) and in silico DNADNA hybridization (isDDH). The clonal relatedness of four E. chengduensis strains and additional 15 ones from NCBI were examined through phylogenetic analysis. The molecular features of E. chengduensis and genetic structure of carbapenemase-encoding plasmids were characterized through genomic annotation and analysis. Results: The results revealed the emergence of blaNDM-1-carrying E. chengduensis strains in China. Multilocus sequence typing (MLST) analysis showed that all 19 E. chengduensis belonged to the same sequence type of ST414. Core SNP analysis suggested the potential intrahospital clonal transmission of ST414 E. chengduensis. The carbapenemase-encoding gene blaNDM-1 was harbored by an IncC-type plasmid, which was experimentally confirmed to be able to conjugate. Discussion: This study reports the first emergence and potential clonal transmission of blaNDM-1-carrying E. chengduensis. Further surveillance should be advocated to monitor the dissemination of carbapenem-resistant E. chengduensis and blaNDM-1-harboring IncC-type plasmids in China. [ABSTRACT FROM AUTHOR]

Currently, antimicrobial resistance (AMR) is a serious health problem in the world, mainly because of the rapid spread of multidrug-resistant (MDR) bacteria. These include bacteria that produce β-lactamases, which confer resistance to β-lactams, the antibiotics with the most prescriptions in the world. Carbapenems are particularly noteworthy because they are considered the ultimate therapeutic option for MDR bacteria. However, this group of antibiotics can also be hydrolyzed by β-lactamases, including metallo-β-lactamases (MBLs), which have one or two zinc ions (Zn2+) on the active site and are resistant to common inhibitors of serine β-lactamases, such as clavulanic acid, sulbactam, tazobactam, and avibactam. Therefore, the design of inhibitors against MBLs has been directed toward various compounds, with groups such as nitrogen, thiols, and metal-binding carboxylates, or compounds such as bicyclic boronates that mimic hydrolysis intermediates. Other compounds, such as dipicolinic acid and aspergillomarasmin A, have also been shown to inhibit MBLs by chelating Zn2+. In fact, recent inhibitors are based on Zn2+ chelation, which is an important factor in the mechanism of action of most MBL inhibitors. Therefore, in this review, we analyzed the current strategies for the design and mechanism of action of metal-ion-binding inhibitors that combat MDR bacteria. [ABSTRACT FROM AUTHOR]

β-lactam resistance is a significant global public health issue. Outbreaks of bacteria resistant to extended-spectrum β-lactams and carbapenems are serious health concerns that not only complicate medical care but also impact patient outcomes. The primary objective of this work was to express and purify two soluble recombinant representative serine β‑lactamases using Escherichia coli strain as an expression host and pET101/D as a cloning vector. Furthermore, a second objective was to evaluate the potential, innovative, and safe use of galloylquinic acid (GQA) from Copaifera lucens as a potential β-lactamase inhibitor. In the present study, blaCTX-M-15 and blaKPC-2 represented genes encoding for serine β-lactamases that were cloned from parent isolates of E. coli and K. pneumoniae, respectively, and expression as well as purification were performed. Moreover, susceptibility results demonstrated that recombinant cells became resistant to all test carbapenems (MICs; 64–128 µg/mL) and cephalosporins (MICs; 128–512 µg/mL). The MICs of the tested β-lactam antibiotics were determined in combination with 4 µg/mL of GQA, clavulanic acid, or tazobactam against E. coli strains expressing CTX-M-15 or KPC-2-β-lactamases. Interestingly, the combination with GQA resulted in an important reduction in the MIC values by 64–512-fold to the susceptible range with comparable results for other reference inhibitors. Additionally, the half-maximal inhibitory concentration of GQA was determined using nitrocefin as a β-lactamase substrate. Data showed that the test agent was similar to tazobactam as an efficient inhibitors of the test enzymes, recording smaller IC50 values (CTX-M-15; 17.51 for tazobactam, 28.16 µg/mL for GQA however, KPC-2; 20.91 for tazobactam, 24.76 µg/mL for GQA) compared to clavulanic acid. Our work introduces GQA as a novel non-β-lactam inhibitor, which interacts with the crucial residues involved in β-lactam recognition and hydrolysis by non-covalent interactions, complementing the enzyme's active site. GQA markedly enhanced the potency of β-lactams against carbapenemase and extended-spectrum β-lactamase-producing strains, reducing the MICs of β-lactams to the susceptible range. The β-lactamase inhibitory activity of GQA makes it a promising lead molecule for the development of more potent β-lactamase inhibitors. [ABSTRACT FROM AUTHOR]

Aims Adequately and accurately identifying carbapenemase-producing Enterobacterales (CPE) is vital for selecting appropriate antimicrobial therapy and implementing effective infection control measures. This study aims to optimize the phenotypic detection method of carbapenemase for routine diagnostics in clinical microbiology laboratories. Methods and Results Carbapenemase genes in 2665 non-duplicate CRE clinical strains collected from various regions of China were confirmed through whole-genome sequencing (WGS). The carbapenemase inhibition test (CIT) was conducted and interpreted using different methods and breakpoints, then compared with the NG-Test CARBA 5 for carbapenemase detection. The diagnostic performance of the CIT method was optimal when the carbapenemase types were determined by comparing the inhibition zone diameters of the imipenem disc with 3-aminophenylboronic acid (APB) plus ethylenediaminetetraacetic acid (EDTA) to those of the imipenem disc with either APB or EDTA alone, with a breakpoint of 4 mm. The overall sensitivities of the current CIT, the modified CIT, and NG-Test CARBA 5 were 91.4%, 94.9%, and 99.9%, respectively. For detecting isolates co-producing Klebsiella pneumoniae carbapenemase (KPC) and metallo-β-lactamases (MBLs), the modified CIT method had higher sensitivity than the current method (70.0% vs. 53.3%), though this difference was not statistically significant (P  = 0.063). The NG-Test CARBA 5 showed excellent performance for multi-carbapenemases diagnosis, with sensitivity and specificity of 97.1% and 100%, respectively. Conclusions Optimizing and standardizing the CIT method for clinical use is necessary. It has certain advantages in diagnosing multi-carbapenemase and rare carbapenemase production. However, for identifying common carbapenemase types, the NG-Test CARBA 5 demonstrated superior performance. [ABSTRACT FROM AUTHOR]

Antibiotic resistance has emerged as a global threat to public health, generating a growing interest in investigating the presence of antibiotic-resistant bacteria in environments influenced by anthropogenic activities. Wastewater treatment plants in hospital serve as significant reservoirs of antimicrobial-resistant bacteria, where a favorable environment is established, promoting the proliferation and transfer of resistance genes among different bacterial species. In our study, we isolated a total of 243 strains from 5 hospital wastewater sites in Mexico, belonging to 21 distinct Gramnegative bacterial species. The presence of β-lactamase was detected in 46.9% (114/243) of the isolates, which belonging to the Enterobacteriaceae family. We identified a total of 169 β-lactamase genes; blaTEM in 33.1%, blaCTX-M in 25.4%, blaKPC in 25.4%, blaNDM 8.8%, blaSHV in 5.3%, and blaOXA-48 in 1.1% distributed in 12 different bacteria species. Among the 114 of the isolates, 50.8% were found to harbor at least one carbapenemase and were discharged into the environment. The carbapenemase blaKPC was found in six Citrobacter spp. and E. coli, while blaNDM was detected in two distinct Enterobacter spp. and E. coli. Notably, blaNDM-1 was identified in a 110 Kb IncFII conjugative plasmid in E. cloacae, E. xiangfangensis, and E. coli within the same hospital wastewater. In conclusion, hospital wastewater showed the presence of Enterobacteriaceae carrying a high frequency of carbapenemase blaKPC and blaNDM. We propose that hospital wastewater serves as reservoirs for resistance mechanism within bacterial communities and creates an optimal environment for the exchange of this resistance mechanism among different bacterial strains. [ABSTRACT FROM AUTHOR]

Antibiotic resistance has become a global threat as it is continuously growing due to the evolution of β‐lactamases diminishing the activity of classic β‐lactam (BL) antibiotics. Recent antibiotic discovery and development efforts have led to the availability of β‐lactamase inhibitors (BLIs) with activity against extended‐spectrum β‐lactamases as well as Klebsiella pneumoniae carbapenemase (KPC)‐producing carbapenem‐resistant organisms (CRO). Nevertheless, there is still a lack of drugs that target metallo‐β‐lactamases (MBL), which hydrolyze carbapenems efficiently, and oxacillinases (OXA) often present in carbapenem‐resistant Acinetobacter baumannii. This review aims to provide a snapshot of microbiology, pharmacology, and clinical data for currently available BL/BLI treatment options as well as agents in late stage development for CRO harboring various β‐lactamases including MBL and OXA‐enzymes. [ABSTRACT FROM AUTHOR]

The emergence of carbapenemase-producing Klebsiella pneumoniae poses a substantial risk to public health. It is essential to comprehend the influence of carbapenemase on the virulence characteristics of K. pneumoniae in order to devise successful strategies for combating these infections. In this study, we explored the distribution disparity of virulence determinants between carbapenemase-producing (CP-Kp, n = 52) and carbapenemase-nonproducing (CN-Kp, n = 43) isolates. The presence of carbapenemases was detected via the modified carbapenem inactivation method and confirmed by PCR. The New Delhi metallo-β-lactamase (blaNDM) and Oxacillinase-48-like (blaOXA-48-like) genes were the most prevalent (94.23% and 76.92%, respectively) in CP-Kp isolates. Coexistence of blaNDM and blaOXA-48-like was observed in 71.15% of isolates, whereas 5.77% coharbored blaNDM and blaKPC. PCR analysis revealed the presence of several virulence genes, including adhesins (fimH, 92.63%, mrkD, 97.89%), capsule-associated virulence (uge, 90.53%), the K2 capsule serotype (k2, 6.32%), the iron acquisition system (kfu, 23.16%), and the regulator of mucoid phenotype (rmpA, 28.42%). A significantly higher prevalence of rmpA was detected in the CP-Kp compared with the CN-Kp (24/52 vs. 3/43, p < 0.0001), indicating a potential association between rmpA and carbapenemase acquisition. In addition, the majority of rmpA (22/24) positive isolates in the CP-Kp isolates coharbored blaNDM and either blaOXA-48-like or blaKPC. [ABSTRACT FROM AUTHOR]

Objective Methods Results Conclusion The global emergence of carbapenem‐resistant Klebsiella pneumoniae is considered a significant contemporary concern., as carbapenems are the last resort for treating infections caused by multidrug‐resistant Gram‐negative bacteria. This study aimed to investigate the carbapenem‐resistance genes in extended‐spectrum β‐lactamase producing K. pneumoniae isolates.Seventy‐five non‐duplicate clinical K. pneumoniae strains were isolated from urine, blood, sputum, and wound samples. Antimicrobial susceptibility tests for 12 different antibiotics were performed using the disk diffusion method, followed by determining minimum inhibitory concentrations of imipenem and meropenem. Phenotypic detection of extended‐spectrum β‐lactamase and carbapenemase enzymes was performed by double‐disc synergy test and modified Hodge test, respectively. PCR assay further investigated resistant isolates for extended‐spectrum β‐lactamase and carbapenemase‐encoding genes.The highest and lowest resistance rates were observed against ampicillin (93.3%) and tigecycline (9.3%). According to phenotypic tests, 46.7% of isolates were positive for extended‐spectrum β‐lactamase enzymes and 52.8% for carbapenemase. A total of 11 isolates contained carbapenemase genes, with blaOXA‐48 (19.4%; 7/36) being the predominant gene, followed by blaNDM (8.3%; 3/36).The study's findings reveal the alarming prevalence of beta‐lactamase enzymes in K. pneumoniae strains. Early detection of carbapenem‐resistant isolates and effective infection control measures are necessary to minimise further spread, as carbapenem resistance has become a public health concern. [ABSTRACT FROM AUTHOR]

The hospital water environment is an important reservoir of multidrug-resistant organisms (MDROs) and presents a risk for patient safety. We assessed the effectiveness of thermal and chemical interventions on sinks contaminated with MDRO in the hospital setting. We conducted a cross-sectional assessment of MDRO contamination of sinks and toilets in 26 clinical wards of a tertiary care hospital. MDRO-contaminated sink traps were then replaced and randomized (1:1:1) to receive chemical (sodium hypochlorite), thermal disinfection (steam), or no intervention. Interventions were repeated weekly for 4 weeks. Sinks were resampled 7 days after the last intervention. The primary outcome was the proportion of decontaminated sinks. MDROs of interest were extended spectrum beta-lactamase (ESBL) producing and carbapenemase-producing Enterobacterales, and non-fermentative Gram-negative bacilli. In the cross-sectional assessment, at least one MDRO was identified in 258 (36%) of the 748 samples and in 91 (47%) of the 192 water sources. In total, 57 (42%) of the 137 sinks and 34 (62%) of the 55 toilets were contaminated with 137 different MDROs. The most common MDRO were ESBL Enterobacterales (69%, 95/137), followed by Verona Integron-Borne Metallo-β-Lactamase (VIM) carbapenemase producing Pseudomonas aeruginosa (9%, 12/137) and Citrobacter spp. (6%, 5/137). In the nested randomized trial, five of the 16 sinks (31%) in the chemical disinfection group were decontaminated, compared with 8 of 18 (44%) in the control group (OR 0.58; 95% CI, 0.14–2.32) and 9 of 17 (53%) in the thermal disinfection group (OR 1.40; 95% CI, 0.37–5.32). Our study failed to demonstrate an added benefit of repeated chemical or thermal disinfection, beyond changing sink traps, in the MDRO decontamination of sinks. Routine chlorine-based disinfection of sinks may need to be reconsidered. [ABSTRACT FROM AUTHOR]

Background: The last-resort classes of antibiotics with the best success in treating severe Pseudomonas aeruginosa (P. aeruginosa) infections caused by drug-resistant P. aeruginosa are carbapenems. So, we aimed to assess the frequency of carbapenemresistant genes in P. aeruginosa. Methods: Samples isolated from critically ill patients at Intensive Care Units (ICUs) and surgical wards in Minia, Egypt were examined for imipenem-resistance phenotypically by Modified Hodge test (MHT) and Modified carbapenem inactivation method (mCIM) test and genotypically by PCR. Results: Fifty isolates (64%) of P. aeruginosa were resistant to carbapenems (imipenem and meropenem), of which: 16 (32%) were (MHT) positive and 26 (52 %) were (mCIM)- positive. Regarding resistance genes, blaIMP, blaVIM, and blaKPC were detected in 14%, 20%, and 48% of the carbapenem resistant Pseudomonas aeruginosa (CRPA) isolates; respectively, while blaGIM, blaSIM, blaNDM-1 and blaSPM-1 were not detected. Conclusion: There is a high prevalence of multi-drug resistant (MDR) strains of P. aeruginosa. Doctors should pay attention to carbapenem resistance. mCIM test was much more sensitive than MHT for phenotypic detection of CRPA isolates. blaKPC gene was the most frequently detected gene. [ABSTRACT FROM AUTHOR]

(1) Background: Carbapenem-resistant (CR) bacteria pose a significant global public health challenge due to their ability to evade treatment with beta-lactam antibiotics, including carbapenems. This study investigates the biofilm-forming capabilities of CR clinical bacterial isolates and examines the impact of serum on biofilm formation. Additionally, the study evaluates the resistance profiles and genetic markers for carbapenemase production. (2) Methods: Bacterial isolates were collected from the microbiology laboratory of Mures County Clinical Hospital between October 2022 and September 2023. Pharyngeal and rectal swabs were screened for carbapenem-resistant bacteria using selective media. Lower respiratory tract samples were also analyzed for CR Gram-negative bacteria. The isolates were tested for their ability to form biofilms in the presence and absence of fetal bovine serum at 24 and 48 h. Carbapenemase production was detected phenotypically and confirmed via PCR for relevant genes. (3) Results: Out of 846 screened samples, 4.25% from pharyngeal swabs and 6.38% from rectal swabs tested positive for CR bacteria. Acinetobacter baumannii and Klebsiella pneumoniae were the most common species isolated. Biofilm formation varied significantly between clinical isolates and standard strains, with clinical isolates generally showing higher biofilm production. The presence of serum had no significant effect on biofilm formation in Klebsiella spp., but stimulated biofilm formation for Acinetobacter spp. Carbapenemase genes blaKPC, blaOXA-48-like, and blaNDM were detected in various isolates, predominantly in Klebsiella spp., but were not the main determinants of carbapenem resistance, at least in screening isolates. (4) Conclusions: This study highlights the variability in biofilm formation among CR clinical isolates and underscores the differences between the bacteria found as carriage versus infection. Both bacterial species and environmental factors variably influence biofilm formation. These insights are crucial for the development of effective treatment and infection control strategies in clinical settings. [ABSTRACT FROM AUTHOR]

Background and Aims: The prevalence of carbapenemase‐producing Enterobacterales (CPE) continues to increase worldwide. Combination of β‐lactam and novel β‐lactamase inhibitors introduce a revolutionary treatment option for CPE. Ceftazidime/avibactam (CAZ/AVB) has been recently developed for treatment of severe infections caused by multidrug‐resistant bacteria. We aimed to evaluate in vitro activity of CAZ/AVB on a collection of 85 ESBL‐producing‐carbapenemase negative and CPE from Iran. Methods: ESBL and carbapenemase production was phenotypically confirmed by combined disk test and modified carbapenem inactivation method respectively. The presence of clinically important carbapenemase encoding genes was examined using PCR. Susceptibility of all isolates to CAZ/AVB was determined using discs containing 30 μg ceftazidime +20 μg avibactam (AVB). Minimum inhibitory concentrations (MICs) of CAZ/AVB in 28 CPE (4 Escherichia coli and 24 Klebsiella pneumoniae) was determined by gradient diffusion method using MIC test strips (0.016−256 mg/L ceftazidime +4 mg/L AVB). Results: All phenotypically identified ESBL positive‐carbapenemase negative isolates were found to be susceptible to CAZ/AVB. Among the carbapenem resistant isolates, CAZ/AVB showed potent inhibitory activity against all OXA‐48‐like (MIC ranges 0.125/4−0.75/4 mg/L) and KPC positive isolates (MIC ranges <0.016/4−0.19/4 mg/L). However, AVB could not restore the activity of ceftazdime against isolates producing metallo‐β‐lactamases (MLBs) including VIM, NDM (MIC > 256/4 mg/L) and IMP (MIC > 8/4 mg/L). Conclusion: Our data highlighted the excellent in vitro performance of CAZ/AVB against ESBL‐producing and CPE suggesting that this combination can efficiently be used as therapeutic option for management of CPE infections particularly in regions with high prevalence of KPC and/or OXA‐48‐like positive but MBL‐negative Enterobacterales. [ABSTRACT FROM AUTHOR]

Background Little is known about the risk of progression from carbapenemase-producing Enterobacterales (CPE) carriage to CPE bloodstream infection (BSI) outside of high-risk settings. We aimed to determine the incidence of CPE BSI among CPE carriers and to assess whether the incidence differs by carbapenemase, species, and setting. Methods We conducted a nationwide population-based retrospective cohort study using national databases. The cohort consisted of all patients in Israel with CPE detected by screening from 1 January 2020 to 10 October 2022. We calculated the cumulative incidence of CPE BSI within 1 year among CPE carriers. We used a competing-risks model with BSI as the outcome and death as the competing risk. Results The study included 6828 CPE carriers. The cumulative incidence of CPE BSI was 2.4% (95% confidence interval [CI], 2.1–2.8). Compared with Klebsiella pneumoniae carbapenemase (KPC), the subhazard of BSI was lower for New Delhi metallo-β-lactamase (NDM) (adjusted subhazard ratio [aSHR], 0.72; 95% CI,.49–1.05) and oxacillinase-48-like (OXA-48-like) (aSHR, 0.60; 95% CI,.32–1.12) but these differences did not reach statistical significance. Compared with K. pneumoniae , the subhazard of BSI was lower for carriers of carbapenemase-producing Escherichia coli (aSHR, 0.33; 95% CI,.21–.52). The subhazard of BSI was higher among patients with CPE carriage first detected in intensive care units (aSHR, 2.10; 95% CI, 1.27–3.49) or oncology/hematology wards (aSHR, 3.95; 95% CI, 2.51–6.22) compared with medical wards. Conclusions The risk of CPE BSI among CPE carriers is lower than previously reported in studies that focused on high-risk patients and settings. The risk of BSI differs significantly by bacterial species and setting, but not by carbapenemase. [ABSTRACT FROM AUTHOR]

Background Klebsiella pneumoniae carbapenemase–producing K pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection. Methods We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct enzyme-linked immunosorbent assay, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model. Results We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In 5 genetically related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsonophagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice. Conclusions Loss of function in wcaJ led to increased complement resistance, complement binding, and opsonophagocytosis, which may promote KPC-Kp persistence by enabling coexistence of increased bloodstream fitness and reduced tissue virulence. [ABSTRACT FROM AUTHOR]

This study was carried out to investigate the biofilm formation capacity of carbapenem resistant P. aeruginosa strains isolated from cattle carcasses by using qualitative and quantitative methods, and also to determine the presence of major carbapenemase and quorum sensing genes. In the study, 13 P. aeruginosa strains, each isolated from a different cattle carcass surface and confirmed by molecular methods, were used as materials. Determination of carbapenem resistance was carried out by disk diffusion method. The presence of blaKPC, blaOXA-48, blaNDM, blaVIM, and blaIMP genes was investigated by PCR. Biofilm production was determined by qualitative and quantitative methods. In order to detect quorum sensing systems, the presence of lasI, lasR, rhlI, and rhlR genes was investigated by PCR. In the study, all P. aeruginosa strains were found to be resistant to at least one of the carbapenems tested. Carbapenemase genes could not be detected in any isolate. All isolates showing carbapenem resistance were found to be quantitatively strong biofilm producers. According to PCR analyses, 12 of the 13 isolates analyzed (92.3%) were found to contain all lasI, lasR, rhlI and rhlR genes. It was determined that one isolate (7.69%) contained only lasR and rhlR genes. With this study, it was determined that carbapenem resistance and biofilm production in P. aeruginosa strains of animal origin are related. [ABSTRACT FROM AUTHOR]

Aims The purpose of this work was to study extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) in freshwaters, hospital effluents, and wastewaters during two sampling campaigns in 2021. Methods and results Water sampling was performed at 24 stations in the Ourthe watershed in Belgium. A total of 644 ESBL (n  = 642) and AmpC (n  = 2) E. coli strains were isolated. Disk-diffusion assays were performed following the EUCAST's recommendations. All strains were tested for the presence of bla CTX-M-1, bla CTX-M-2, and bla CTX-M-9 gene groups by PCR. Genes belonging to bla CTX-M-1 and bla CTX-M-9 groups were detected, respectively, in 73.6% and 14.9% of the strains. No bla CTX-M-2 group's gene was found. A subset of strains (n  = 40) was selected for whole genome sequencing. Escherichia coli serotype O18: H7 ST 1463 was predominant (n  = 14) in the sequenced strains and showed pathogenicity in the Galleria mellonella larvae model. β-lactamase genes identified were bla CTX-M (n  = 21), with bla CTX-M-15 mostly represented (n  = 15), as well as bla TEM (n  = 11), bla OXA (n  = 7), bla SHV (n  = 9), and carbapenemase (CP) genes were observed in several strains— bla KPC-3 (n  = 19), bla NDM-1 (n  = 1), bla VIM-1 (n  = 2), and bla OXA-244 (n  = 2)—even from freshwaters. Conclusions ESBL-EC are widely distributed in the aquatic environment in Belgium and contain a variety of ESBL and CP genes. [ABSTRACT FROM AUTHOR]

Purpose: The current study evaluated the in vitro activities of ceftolozane/tazobactam (C/T), imipenem/relebactam (IMI/REL), and comparators against recent (2017–2021) clinical isolates of gram-negative bacilli from two countries in southern Europe. Methods: Nine clinical laboratories (two in Greece; seven in Italy) each collected up to 250 consecutive gram-negative isolates per year from lower respiratory tract, intraabdominal, urinary tract, and bloodstream infection samples. MICs were determined by the CLSI broth microdilution method and interpreted using 2022 EUCAST breakpoints. β-lactamase genes were identified in select β-lactam-nonsusceptible isolate subsets. Results: C/T inhibited the growth of 85–87% of Enterobacterales and 94–96% of ESBL-positive non-CRE NME (non-Morganellaceae Enterobacterales) isolates from both countries. IMI/REL inhibited 95–98% of NME, 100% of ESBL-positive non-CRE NME, and 98–99% of KPC-positive NME isolates from both countries. Country-specific differences in percent susceptible values for C/T, IMI/REL, meropenem, piperacillin/tazobactam, levofloxacin, and amikacin were more pronounced for Pseudomonas aeruginosa than Enterobacterales. C/T and IMI/REL both inhibited 84% of P. aeruginosa isolates from Greece and 91–92% of isolates from Italy. MBL rates were estimated as 4% of Enterobacterales and 10% of P. aeruginosa isolates from Greece compared to 1% of Enterobacterales and 3% of P. aeruginosa isolates from Italy. KPC rates among Enterobacterales isolates were similar in both countries (7–8%). OXA-48-like enzymes were only identified in Enterobacterales isolates from Italy (1%) while GES carbapenemase genes were only identified in P. aeruginosa isolates from Italy (2%). Conclusion: We conclude that C/T and IMI/REL may provide viable treatment options for many patients from Greece and Italy. [ABSTRACT FROM AUTHOR]

The emergence of extensively-resistant strains of Klebsiella pneumoniae (K. pneumoniae) in healthcare settings is linked to prolonged hospitalization and uncontrolled use of antibiotics. There is a paucity of data regarding the prevalence and mechanisms of colistin and fosfomycin resistance encoding genes rate and mechanisms in Iran. The objective of this study was to determine the prevalence of biofilm formation and fosfomycin and colistin resistance among K. pneumoniae strains producing ESBL and carbapenemases by detecting the mcr-1, mcr-2, and fosA genes in Tehran, Iran, during the 2020-2021 period. After collecting 73 samples, the isolates were identified using biochemical tests. Antibiotic susceptibility test was performed using the disk diffusion method. The phenotypic determination of extended-spectrum beta-lactamases (ESBLs) and carbapenemase enzymes was conducted using combined disk and CARBA-NP tests, respectively. The biofilm formation was conducted using a microtiter tissue plate assay. Polymerase chain reaction (PCR) was employed to detect the mcr-1, mcr-2 and fosA genes, which are associated with colistin and fosfomycin resistance, respectively. The highest resistance rate was observed against ampicillin (97%), chloramphenicol (90%), and ciprofloxacin (87%), respectively. In contrast, the lowest resistance rate was noted against gentamicin (4%), amikacin (10%), and cotrimoxazole (18%). Moreover, 44 and 23 isolates were identified as ESBL and carbapenemase -producing K. pneumonia), respectively. Of the fortyeight isolates that formed strong biofilms, one was a non-biofilm producer. The PCR test revealed the amplification of the fosA2 gene in four isolates and the mcr-2 genes in one isolate. However, no amplification of the fosA3 or mcr-1 genes was observed. The present study demonstrated that the frequency of K. pneumoniae isolates producing ESBL and carbapenemase, as well as mcr-1, mcr-2 and fosA genes, was relatively low. However, given the potential for these genes to be disseminated more widely, it is imperative to implement effective isolation and control measures. Moreover, these strains demonstrated the capacity to form biofilms in vitro, which can lead to persistent infections in the hospital settings. [ABSTRACT FROM AUTHOR]

OXA-244, an R214G variant of OXA-48, is silently spreading worldwide likely because of difficulties in detection using classical screening media. Here, we characterized two clinical isolates of Escherichia coli and Citrobacter youngae that displayed reduced susceptibility to carbapenems but were lacking significant carbapenemase activity as revealed by negative Carba NP test results. However, positive test results were seen for OXA-48-like enzymes by lateral flow immunoassays. WGS revealed the presence of a blaOXA-181-like gene that codes for OXA-484, an R214G variant of OXA-181. BlaOXA-484 gene was located on a 58.4-kb IncP1-like plasmid (pN-OXA-484), that upon transfer into E. coli HB4 with impaired permeability, conferred carbapenem and temocillin resistance (MICs > 32 mg/L). E. coli TOP10 (pTOPO-OXA-484) revealed reduced MICs in most substrates as compared to E. coli TOP10 (pTOPO-OXA-181), especially for imipenem (0.25 mg/L versus 0.75 mg/L) and temocillin (16 mg/L versus 1028 mg/L). Catalytic efficiencies of OXA-484 were reduced as compared to OXA-181 for most ß-lactams including imipenem and temocillin with 27.5- and 21.7-fold reduction, respectively. Molecular modeling confirmed that the salt bridges between R214, D159, and the R1 substituent's carboxylate group of temocillin were not possible with G214 in OXA-484, explaining the reduced affinity for temocillin. In addition, changes in active site's water network may explain the decrease in hydrolysis rate of carbapenems. OXA-484 has weak imipenem and temocillin hydrolytic activities, which may lead to silent spread due to underdetection using selective screening media or biochemical imipenem hydrolysis confirmatory tests. [ABSTRACT FROM AUTHOR]

Introduction: This study aimed to investigate the emergence and characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains that demonstrate resistance to multiple antibiotics, including aminoglycosides and tigecycline, in a Chinese hospital. Methods: A group of ten CRKP strains were collected from the nine patients in a Chinese hospital. Antimicrobial Susceptibility Testing (AST) and phenotypic inhibition assays precisely assess bacterial antibiotic resistance. Real-time quantitative PCR (RT-qPCR) was used to analyze the mRNA levels of efflux pump genes (acrA/acrB and oqxA/oqxB) and the regulatory gene (ramA). The core-genome tree and PFGE patterns were analyzed to assess the clonal and horizontal transfer expansion of the strains. Whole-genome sequencing was performed on a clinical isolate of K. pneumoniae named Kpn20 to identify key resistance genes and antimicrobial resistance islands (ARI). Results: The CRKP strains showed high resistance to carbapenems, aminoglycosides (CLSI, 2024), and tigecycline (EUCAST, 2024). The mRNA expression levels of efflux pump genes and regulatory genes were detected by RT-qPCR. All 10 isolates had significant differences compared to the control group of ATCC13883. The core-genome tree and PFGE patterns revealed five clusters, indicating clonal and horizontal transfer expansion. Three key resistance genes (blaoxa-232, blaCTX-M-15, and rmtF) were observed in the K. pneumoniae clinical isolate Kpn20. Mobile antibiotic resistance islands were identified containing blaCTX-M-15 and rmtF, with multiple insertion sequences and transposons present. The coexistence of blaoxa-232 and rmtF in a high-risk K. pneumoniae strain was reported. Conjugation assay was utilized to investigate the transferability of blaoxa-232-encoding plasmids horizontally. Conclusion: The study highlights the emergence of ST15-KL112 high-risk CRKP strains with multidrug resistance, including to aminoglycosides and tigecycline. The presence of mobile ARI and clonal and horizontal transfer expansion of strains indicate the threat of transmission of these strains. Future research is needed to assess the prevalence of such isolates and develop effective control measures. [ABSTRACT FROM AUTHOR]

Objectives: The emergence of multidrug-resistant (MDR) Acinetobacter baumannii has become a serious worldwide medical problem. This study was designed to clarify the genetic and epidemiological properties of MDR A. baumannii clinical isolates. Methods: A total of 66 MDR A. baumannii isolates were obtained from 66 inpatients between May 2019 and February 2020 in a university hospital in Nepal. Whole genomes of these isolates were sequenced using next-generation sequencing. Phylogenetic trees were constructed from single nucleotide polymorphism concatemers. Multilocus sequence typing (MLST) and clonal complex (CC) analysis were conducted, and drug-resistance genes were identified. Results: Of the 66 isolates, 26 harboured a gene encoding NDM-type metallo-β-lactamase, and 55 harboured a gene encoding the 16S rRNA methyltransferase, ArmA. All isolates had point mutations in the quinolone-resistance-determining regions of gyrA and parC. Phylogenetic analysis showed that 55 isolates harboured armA, 26 harboured blaNDM-1, and14 harboured blaPER-7. Multilocus sequence typing and CC analysis revealed that 34 isolates belonged to CC2 (ST2), 10 to CC1 (nine ST1 and one ST623), and eight to CC149 (ST149). Compared to our previous study on MDR A. baumannii in Nepal in 2012, the isolation rate of CC2 increased, whereas that of CC149 decreased between 2012 and 2020. Conclusions: This study indicates that MDR A. baumannii producing carbapenemase and 16S rRNA methyltransferase, with high resistance to carbapenems and/or aminoglycosides, are spreading in medical settings in Nepal. The genetic backgrounds of MDR A. baumannii isolates have shifted to international clone 2 over several years.

Objective: Multidrug-resistant (MDR) Acinetobacter baumannii is a major issue within healthcare facilities in Bangladesh due to its frequent association with hospital-acquired infections. In this study we report on a carbapenem-resistant draft genome sequence of an A. baumannii BD20 sample isolated from an infected wound in Bangladesh. Methods: A. baumannii BD20 was isolated from an infected burn wound. Whole-genome sequencing was carried out and annotated using PGAP and Prokka. Sequence type, antimicrobial resistance genes, virulence factor genes, and metal resistance genes were investigated. Core genome multilocus sequence typing–based phylogenomic analysis between A. baumannii BD20 and 213 A. baumannii strains retrieved from the NCBI GenBank database was performed using the BacWGSTdb 2.0 server. Results: A. baumannii BD20 (MLST 491) was resistant to all the antibiotics tested, except for colistin and polymyxin B. Along with many other antibiotic resistance genes, the isolate harbored three classes of beta lactamase–producing genes: blaGES-11 (class A), blaOXA-69 (class D), blaADC-10 (class C), and blaADC-11 (class C). Additionally, the strain carried several virulence genes and metal resistance determinants, which may contribute to its increased virulence. Core genome MLST–based phylogenomic analysis revealed that A. baumannii BD20 was closely related to another ST491 strain isolated from Singapore. Conclusions: The findings of this study underscore the growing challenge of MDR A. baumannii, emphasizing the need for vigilant surveillance and infection-control measures in healthcare settings in order to address these emerging threats effectively.

Objectives: The aim of this study was to characterize an NDM-1-producing Acinetobacter seifertii isolates from a patient in South Korea. Methods: Antibiotic susceptibility testing and genotyping using multigene sequencing were performed and whole plasmid sequences were determined. Results: The genotype of A. seifertii was ST1899 and was resistant to ceftazidime, trimethoprim–sulfamethoxazole, and piperacillin–tazobactam, in addition to carbapenem. blaNDM-1 was surrounded by the ISAba125 insertion sequence within the structure of Tn125 in the 47 kb-sized plasmid. The plasmid exhibited a structure similar to that of other plasmids of diverse Acinetobacter sp. found worldwide. Transconjugation and the growth curve indicated that the plasmid was adapted to A. seifertii rather than other closely related Acinetobacter sp. Conclusions: Acquisition of carbapenem resistance by horizontal transfer of the blaNDM-1-carrying plasmid from another Acinetobacter species was found with no growth defect.

Abstract β-lactam resistance is a significant global public health issue. Outbreaks of bacteria resistant to extended-spectrum β-lactams and carbapenems are serious health concerns that not only complicate medical care but also impact patient outcomes. The primary objective of this work was to express and purify two soluble recombinant representative serine β‑lactamases using Escherichia coli strain as an expression host and pET101/D as a cloning vector. Furthermore, a second objective was to evaluate the potential, innovative, and safe use of galloylquinic acid (GQA) from Copaifera lucens as a potential β-lactamase inhibitor. In the present study, bla CTX-M-15 and bla KPC-2 represented genes encoding for serine β-lactamases that were cloned from parent isolates of E. coli and K. pneumoniae, respectively, and expression as well as purification were performed. Moreover, susceptibility results demonstrated that recombinant cells became resistant to all test carbapenems (MICs; 64–128 µg/mL) and cephalosporins (MICs; 128–512 µg/mL). The MICs of the tested β-lactam antibiotics were determined in combination with 4 µg/mL of GQA, clavulanic acid, or tazobactam against E. coli strains expressing CTX-M-15 or KPC-2-β-lactamases. Interestingly, the combination with GQA resulted in an important reduction in the MIC values by 64–512-fold to the susceptible range with comparable results for other reference inhibitors. Additionally, the half-maximal inhibitory concentration of GQA was determined using nitrocefin as a β-lactamase substrate. Data showed that the test agent was similar to tazobactam as an efficient inhibitors of the test enzymes, recording smaller IC50 values (CTX-M-15; 17.51 for tazobactam, 28.16 µg/mL for GQA however, KPC-2; 20.91 for tazobactam, 24.76 µg/mL for GQA) compared to clavulanic acid. Our work introduces GQA as a novel non-β-lactam inhibitor, which interacts with the crucial residues involved in β-lactam recognition and hydrolysis by non-covalent interactions, complementing the enzyme’s active site. GQA markedly enhanced the potency of β-lactams against carbapenemase and extended-spectrum β-lactamase-producing strains, reducing the MICs of β-lactams to the susceptible range. The β-lactamase inhibitory activity of GQA makes it a promising lead molecule for the development of more potent β-lactamase inhibitors.

This work explores the potential of a triple combination of meropenem (MEM), a novel metallo-β-lactamase (MBL) inhibitor (indole-2-carboxylate 58 (InC58)), and a serine-β-lactamase (SBL) inhibitor (avibactam (AVI)) for broad-spectrum activity against carbapenemase-producing bacteria. A diverse panel comprising MBL- and SBL-producing strains was used for susceptibility testing of the triple combination using the agar dilution method. The frequency of resistance (FoR) to MEM combined with InC58 was investigated. Mutants were sequenced and tested for cross resistance, fitness, and the stability of the resistance phenotype. Compared with the double combinations of MEM plus an SBL or MBL inhibitor, the triple combination extended the spectrum of activity to most of the isolates bearing SBLs (oxacillinase-48 (OXA-48) and Klebsiella pneumoniae carbapenemase-2 (KPC-2)) and MBLs (New Delhi metallo-β-lactamases (NDMs)), although it was not effective against Verona integron-encoded metallo-β-lactamase (VIM)-carrying Pseudomonas aeruginosa (P. aeruginosa) and OXA-23-carrying Acinetobacter baumannii (A. baumannii). The FoR to MEM plus InC58 ranged from 2.22 × 10−7 to 1.13 × 10−6. The resistance correlated with mutations to ompC and comR, affecting porin C and copper permeability, respectively. The mutants manifested a fitness cost, a decreased level of resistance during passage without antibiotic pressure, and cross resistance to another carbapenem (imipenem) and a β-lactamase inhibitor (taniborbactam). In conclusion, compared with the dual combinations, the triple combination of MEM with InC58 and AVI showed a much wider spectrum of activity against different carbapenemase-producing bacteria, revealing a new strategy to combat β-lactamase-mediated antimicrobial resistance.

Bailong Hou,1,&ast; Ying Zhou,2,&ast; Wei Wang,1 Weifeng Shen,1 Qinlong Yu,1 Minjie Mao,1 Siheng Wang,1 Wenxiu Ai,3 Fangyou Yu,2,3 Pingyang Shao1 1Department of Clinical Laboratory Medicine, The First Hospital of Jiaxing, The Affiliated Hospital of Jiaxing University, Jiaxing, 314000, People’s Republic of China; 2Department of Clinical Laboratory Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, 200000, People’s Republic of China; 3Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Pingyang Shao, Department of Clinical Laboratory Medicine, The First Hospital of Jiaxing, The Affiliated Hospital of Jiaxing University, Jiaxing, 314000, People’s Republic of China, Email spy202001@163.com Fangyou Yu, Department of Clinical Laboratory Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, 200000, People’s Republic of China, Email wzjxyfy@163.comIntroduction: This study aimed to investigate the emergence and characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains that demonstrate resistance to multiple antibiotics, including aminoglycosides and tigecycline, in a Chinese hospital.Methods: A group of ten CRKP strains were collected from the nine patients in a Chinese hospital. Antimicrobial Susceptibility Testing (AST) and phenotypic inhibition assays precisely assess bacterial antibiotic resistance. Real-time quantitative PCR (RT-qPCR) was used to analyze the mRNA levels of efflux pump genes (acrA/acrB and oqxA/oqxB) and the regulatory gene (ramA). The core-genome tree and PFGE patterns were analyzed to assess the clonal and horizontal transfer expansion of the strains. Whole-genome sequencing was performed on a clinical isolate of K. pneumoniae named Kpn20 to identify key resistance genes and antimicrobial resistance islands (ARI).Results: The CRKP strains showed high resistance to carbapenems, aminoglycosides (CLSI, 2024), and tigecycline (EUCAST, 2024). The mRNA expression levels of efflux pump genes and regulatory genes were detected by RT-qPCR. All 10 isolates had significant differences compared to the control group of ATCC13883. The core-genome tree and PFGE patterns revealed five clusters, indicating clonal and horizontal transfer expansion. Three key resistance genes (blaoxa-232, blaCTX-M-15, and rmtF) were observed in the K. pneumoniae clinical isolate Kpn20. Mobile antibiotic resistance islands were identified containing blaCTX-M-15 and rmtF, with multiple insertion sequences and transposons present. The coexistence of blaoxa-232 and rmtF in a high-risk K. pneumoniae strain was reported. Conjugation assay was utilized to investigate the transferability of blaoxa-232-encoding plasmids horizontally.Conclusion: The study highlights the emergence of ST15-KL112 high-risk CRKP strains with multidrug resistance, including to aminoglycosides and tigecycline. The presence of mobile ARI and clonal and horizontal transfer expansion of strains indicate the threat of transmission of these strains. Future research is needed to assess the prevalence of such isolates and develop effective control measures.Keywords: blaOXA-232, rmtF, Klebsiella pneumoniae, carbapenemase, plasmid

Introduction: Infections caused by Klebsiella pneumoniae carbapenemase (KPC) pose a significant clinical challenge due to increasing antibiotic resistance. This study analyzes the effectiveness of ceftazidime/avibactam treatment for KPC infections, focusing on 30-day mortality and comparing results with control groups treated with different antibiotics. Material and methods: We conducted a retrospective analysis of six cohort studies, comparing thirty-day mortality in patients treated with ceftazidime/avibactam with control groups using other antibiotics. The studies included patients infected with KPC, and the analysis focused on therapeutic effectiveness. Aim of the study: The aim of the study is to compare the results in terms of 30-day mortality in groups treated with ceftazidime/avibactam compared to control groups using different antibiotics. Conclusions: Data analysis from various studies revealed varied 30-day mortality outcomes in groups treated with ceftazidime/avibactam compared to control groups using different antibiotics. Ceftazidime/avibactam proved to be more effective in all studies, reducing mortality rates compared to other treatment regimens. We emphasize that emerging antibiotic resistance, especially in the case of KPC, requires a comprehensive therapeutic approach. Despite promising ceftazidime/avibactam results, factors such as overall patient health and treatment delays may influence final therapy outcomes. We also highlight controversies regarding combination therapy vs. monotherapy, necessitating further research. Our work underscores the importance of monitoring the effectiveness of KPC infection treatment and exploring new therapeutic strategies. Further clinical studies are essential to develop a fuller understanding and optimal therapeutic protocols in the face of the growing antibiotic resistance problem. [ABSTRACT FROM AUTHOR]

The carbapenem-resistant Enterobacterales (CRE) pose a pressing public health concern. Here, we investigated the frequency of CRE bacteria, carbapenemase-encoding genes, and the molecular epidemiology of carbapenemase-resistant Escherichia coli in wastewater resources and healthy carriers in Iran. Out of 617 Enterobacterales bacteria, 24% were carbapenem-resistant. The prevalence of CRE bacteria in livestock and poultry wastewater at 34% and hospital wastewater at 33% was significantly higher (P ≤ 0.05) than those in healthy carriers and municipal wastewater at 22 and 17%, respectively. The overall colonization rate of CRE in healthy individuals was 22%. Regarding individual Enterobacterales species, the following percentages of isolates were found to be CRE: E. coli (18%), Citrobacter spp. (24%), Klebsiella pneumoniae (28%), Proteus spp. (40%), Enterobacter spp. (25%), Yersinia spp. (17%), Hafnia spp. (31%), Providencia spp. (21%), and Serratia spp. (36%). The blaOXA-48 gene was detected in 97% of CRE isolates, while the blaNDM and blaVIM genes were detected in 24 and 3% of isolates, respectively. The B2 phylogroup was the most prominent group identified in carbapenem-resistant E. coli isolates, accounting for 80% of isolates. High prevalence of CRE with transmissible carbapenemase genes among healthy people and wastewater in Iran underscores the need for assertive measures to prevent further dissemination. HIGHLIGHTS Twenty-four percent of isolates, including nine species, were carbapenem-resistant.; CRE bacteria were widely distributed in various wastewater resources and healthy individuals.; CRE bacteria were more prevalent in hospital, livestock, and poultry wastewater.; The blaOXA-48 gene was detected in 97% of CRE isolates, followed by blaNDM, 24%, and blaVIM, 3%.; Carbapenem-resistant E. coli isolates mainly belonged to the B2 phylogroup.;

Weihua Han,1,&ast; Peiyao Zhou,2,&ast; Chun Chen,3,&ast; Chunyang Wu,2 Li Shen,1 Cailing Wan,4 Yanghua Xiao,4 Jiao Zhang,2 Bingjie Wang,1 Junhong Shi,1 Xinru Yuan,1 Haojin Gao,1 Hongxiu Wang,1 Ying Zhou,1 Fangyou Yu1 1Department of Clinical Laboratory, Shanghai Pulmonary Hospital, School of Medicine, Tongji University, Shanghai, People’s Republic of China; 2Department of Laboratory Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, People’s Republic of China; 3Cancer Center, Department of Pulmonary and Critical Care Medicine, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, Hangzhou, People’s Republic of China; 4School of Public Health, Nanchang University, Nanchang, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Ying Zhou; Fangyou Yu, Email 18702195157@163.com; wzjxyfy@163.comBackground: Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are a great threat to public health worldwide. Ceftazidime-avibactam (CZA) is an effective β-lactam/β-lactamase inhibitors against CRKP. However, reports of resistance to CZA, mainly caused by Klebsiella pneumoniae carbapenemase (KPC) variants, have increased in recent years. In this study, we aimed to describe the resistance characteristics of KPC-12, a novel KPC variant identified from a CZA resistant K. pneumoniae.Methods: The K. pneumoniae YFKP-97 collected from a patient with respiratory tract infection was performed whole-genome sequencing (WGS) on the Illumina NovaSeq 6000 platform. Genomic characteristics were analyzed using bioinformatics methods. Antimicrobial susceptibility testing was conducted by the broth microdilution method. Induction of resistant strain was carried out in vitro as previously described. The G. mellonella killing assay was used to evaluate the pathogenicity of strains, and the conjugation experiment was performed to evaluate plasmid transfer ability.Results: Strain YFKP-97 was a multidrug-resistant clinical ST11-KL47 K. pneumoniae confers high-level resistance to CZA (16/4 μg/mL). WGS revealed that a KPC variant, KPC-12, was carried by the IncFII (pHN7A8) plasmids (pYFKP-97_a and pYFKP-97_b) and showed significantly decreased activity against carbapenems. In addition, there was a dose-dependent effect of blaKPC-12 on its activity against ceftazidime. In vitro inducible resistance assay results demonstrated that the KPC-12 variant was more likely to confer resistance to CZA than the KPC-2 and KPC-3 variants.Discussion: Our study revealed that patients who was not treated with CZA are also possible to be infected with CZA-resistant strains harbored a novel KPC variant. Given that the transformant carrying blaKPC-12 was more likely to exhibit a CZA-resistance phenotype. Therefore, it is important to accurately identify the KPC variants as early as possible.Keywords: carbapenem-resistant Enterobacterales, Klebsiella pneumoniae, ceftazidime-avibactam, KPC-12, carbapenemase

We characterized a collection of IMI-like–producing Enterobacter spp. isolates (n = 112) in France. The main clone corresponded to IMI-1–producing sequence type 820 E. cloacae subspecies cloacae that was involved in an outbreak. Clinicians should be aware of potential antimicrobial resistance among these bacteria.

Abstract Background Aztreonam-avibactam (ATM-AVI) combination shows promising effectiveness on most carbapenemase-producing Gram-negatives, yet standardized antibiotic susceptibility testing (AST) methods for evaluating the combination in clinical laboratories is lacking. We aimed to evaluate different ATM-AVI AST approaches. Methods 96 characterized carbapenem-resistant clinical isolates belonging to 9 Enterobacterales (EB; n = 80) and P. aeruginosa (PA; n = 16) species, including 90 carbapenemase producers and 72 strains resistant to both CAZ-AVI and ATM, were tested. Paper disk elution (DE; Bio-Rad) and E-test gradient strips stacking (SS; bioMérieux) were performed for the ATM + CAZ-AVI combination. MIC Test Strip (MTS; Liofilchem) was evaluated for ATM-AVI MIC determination. Results were interpreted applying ATM clinical breakpoints of the EUCAST guidelines and compared to the broth microdilution method (Sensititre, Thermofisher). Results According to broth microdilution method, 93% of EB and 69% of PA were tested susceptible to ATM-AVI. The synergistic effect of ATM-AVI was of 95% for EB, but of only 17% for PA. The MTS method yielded higher categorical and essential agreement (CA/EA) rates for both EB (89%/91%) and PA (94%/94%) compared to SS, where the rates were 87%/83% for EB and 81%/81% for PA. MTS and SS yielded 2 and 3 major discrepancies, respectively, while 3 very major discrepancies each were observed for both methods. Concerning the DE method, CA reached 91% for EB and 81% for PA, but high number of very major discrepancies were observed for EB (n = 6; 8%) and for PA (n = 3; 19%). Conclusions The ATM-AVI association displayed excellent in vitro activity against highly resistant clinical Enterobacterales strains. MTS method offers accurate ATM-AVI AST results, while the SS method might serve as better alternative then DE method in assessing the efficacy of ATM + CAZ-AVI combination. However, further investigation is needed to confirm the methods' ability to detect ATM-AVI resistance.

Abstract Antimicrobial resistance is considered one of the most critical threat for both human and animal health. Recently, reports of infection or colonization by carbapenemase-producing Enterobacterales in companion animals had been described. This study report the first molecular characterization of NDM-producing Enterobacterales causing infections in companion animals from Argentina. Nineteen out of 3662 Enterobacterales isolates analyzed between October 2021 and July 2022 were resistant to carbapenemes by VITEK2C and disk diffusion method, and suspected to be carbapenemase-producers. Ten isolates were recovered from canine and nine from feline animals. Isolates were identified as K. pneumoniae (n = 9), E. coli (n = 6) and E. cloacae complex (n = 4), and all of them presented positive synergy among EDTA and carbapenems disks, mCIM/eCIM indicative of metallo-carbapenemase production and were also positive by PCR for bla NDM gene. NDM variants were determined by Sanger sequencing method. All 19 isolates were resistant to β-lactams and aminoglycosides but remained susceptible to colistin (100%), tigecycline (95%), fosfomycin (84%), nitrofurantoin (63%), minocycline (58%), chloramphenicol (42%), doxycycline (21%), enrofloxacin (5%), ciprofloxacin (5%) and trimethoprim/sulfamethoxazole (5%). Almost all isolates (17/19) co-harbored bla CTX-M plus bla CMY, one harbored bla CTX-M alone and the remaining bla CMY. E. coli and E. cloacae complex isolates harbored bla CTX-M-1/15 or bla CTX-M-2 groups, while all K. pneumoniae harbored only bla CTX-M-1/15 genes. All E. coli and E. cloacae complex isolates harbored bla NDM-1, while in K. pneumoniae bla NDM-1 (n = 6), bla NDM-5 (n = 2), and bla NDM-1 plus bla NDM-5 (n = 1) were confirmed. MLST analysis revealed the following sequence types by species, K. pneumoniae: ST15 (n = 5), ST273 (n = 2), ST11, and ST29; E. coli: ST162 (n = 3), ST457, ST224, and ST1196; E. cloacae complex: ST171, ST286, ST544 and ST61. To the best of our knowledge, this is the first description of NDM-producing E. cloacae complex isolates recovered from cats. Even though different species and clones were observed, it is remarkable the finding of some major clones among K. pneumoniae and E. coli, as well as the circulation of NDM as the main carbapenemase. Surveillance in companion pets is needed to detect the spread of carbapenem-resistant Enterobacterales and to alert about the dissemination of these pathogens among pets and humans.

Pseudomonas aeruginosa is commonly associated with the ability to acquire antimicrobial resistance. The surveillance of resistance genes in various environmental matrices has gained prominence in recent years, being seen as a potential threat to public health. The objective of this study was to investigate genes encoding metallo-beta-lactamases (MBLs), which confer resistance to carbapenems, in wastewater. Fifteen isolates of P. aeruginosa were collected for five months from samples obtained from a municipal wastewater treatment plant in Rio Grande do Sul. These isolates were subjected to disk diffusion testing using 10 different antimicrobials. Phenotypic enzymatic tests for MBLs were conducted, and positive isolates underwent DNA extraction and gene detection using the polymerase chain reaction. The resistance rate to ceftazidime was 100%, cefepime 73.3%, piperacillin–tazobactam 66.67%, imipenem 53.30%, levofloxacin 46.67%, tobramycin 40%, and ciprofloxacin and amikacin 13.33%. Both meropenem and aztreonam resistances were rare accounting for 6.60% of the tested isolates. Among these isolates, 20% were classified as multidrug-resistant and were found to carry the blaNDM and blaSPM genes. The results suggest that evaluating resistance genes in bacteria from urban raw sewage can provide data that assist in surveillance, as this environment can stimulate increased bacterial resistance. HIGHLIGHTS Twenty per cent of P. aeruginosa isolates were classified as multidrug-resistant (MDR).; Urban sewage, which is associated with hospital sewage contributes to the spread of antimicrobial resistance genes (ARGs).; Three isolates carrying genes encoding MBLs presented resistance to beta-lactam antibiotics.; P. aeruginosa showed resistance characteristics to different classes of antibiotics and may have suffered stress of anthropogenic origin.;

Il Kwon Bae, Jun Sung Hong Department of Companion Animal Health and Science, Silla University, Busan, South KoreaCorrespondence: Jun Sung Hong, Department of Companion Animal Health and Science, Silla University, 140 Baegyang-Daero (Blvd), 700beon-Gil Road, Sasang-Gu, Busan, 46958, South Korea, Email jumphong2@nate.comBackground: Clinical isolates of Acinetobacter species in South Korea are continuously exhibiting high rates of antimicrobial resistance to carbapenems, indicating that there are public health concerns among both healthcare-associated infections and community-associated infections. The aim of this study was to describe the prevalence and characteristics of carbapenem-resistant Acinetobacter isolates originating from community hospitals.Materials and Methods: A total of 817 non-duplicated Acinetobacter species were isolated from December 2022 to July 2023 at long-term care facilities and general hospitals in 16 regions geographically distributed throughout South Korea. Bacterial identification and antimicrobial susceptibility testing were performed using the VITEK-2 system. The bacteria were identified as Acinetobacter baumannii by blaOXA-51 PCR and as non-baumannii Acinetobacter species by rpoB sequence analysis. The carbapenem resistance genes (OXA-23, OXA-48, OXA-58, IMP, VIM, NDM, GES, and KPC) were identified via PCR and sequencing. The genetic relatedness of carbapenem-resistant A. baumannii (CRAB) isolates was assessed by multilocus sequence typing.Results: A total of 659 A. baumannii and 158 non-baumannii Acinetobacter isolates, comprising 19 different species, were identified in all 16 regions. The carbapenem resistance rate was 87.4% (n=576) for the A. baumannii isolates, and all the strains produced blaOXA-23. For non-baumannii Acinetobacter, the rate of carbapenem resistance was 8.9% (n=14); this resistance was primarily caused by blaOXA-23 (n=9), followed by blaNDM-1 (n=3) and blaVIM-2 (n=2). Of the 576 CRAB isolates, clonal complex 92 (CC92) was the predominant genotypes, followed by sequence type 229 (ST229), ST373, ST397, ST447, and ST620.Conclusion: Our results showed the distribution of Acinetobacter species and showed that CC92 CRAB clinical isolates with widespread production of blaOXA-23 were predominant in community hospitals. Our findings suggest that there is a need for urgent and effective methods to reduce carbapenem resistance in A. baumannii in South Korea.Keywords: Acinetobacter, carbapenem, carbapenemase, OXA-23, CC92, community hospitals

Abstract Background Klebsiella pneumoniae infections have become a major cause of hospital acquired infection worldwide with the increased rate of acquisition of resistance to antibiotics. Carbapenem resistance mainly among Gram negative is an ongoing problem which causes serious outbreaks dramatically limiting treatment options. This prospective cross-sectional study was designed to detect bla KPC gene from carbapenem resistant K. pneumoniae. Materials and Methods A totally of 1118 different clinical specimens were screened and confirmed for KPC producing K. pneumoniae phenotypically using Meropenem (10 μg) disc. The bla KPC gene was amplified from the isolates of K. pneumoniae to detect the presence of this gene. Result Of the total samples processed, 18.6% (n = 36) were K. pneumoniae and among 36 K. pneumoniae, 61.1% (n = 22/36) were meropenem resistant. This study demonstrated the higher level of MDR 91.7% (n = 33) and KPC production 47.2% (n = 17) among K. pneumoniae isolates. The bla KPC gene was detected in 8.3% (n = 3) of meropenem resistant isolates. Conclusion Since the study demonstrates the higher level of MDR and KPC producing K. pneumoniae isolates that has challenged the use of antimicrobial agents, continuous microbiology, and molecular surveillance to assist early detection and minimize the further dissemination of bla KPC should be initiated. We anticipate that the findings of this study will be useful in understanding the prevalence of KPC-producing K. pneumoniae in Nepal.

Huaming Xu,1 Chunhui Lin,2,3 Hao Tang,4 Rongrong Li,2,3 Zhaoxin Xia,2,3 Yi Zhu,3 Zhen Liu,2,3 Jilu Shen2,3 1The First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei, People’s Republic of China; 2The First Affiliated Hospital of Anhui Medical University, Hefei, People’s Republic of China; 3Anhui Public Health Clinical Center, Hefei, People’s Republic of China; 4The Second Affiliated Hospital of Anhui Medical University, Hefei, People’s Republic of ChinaCorrespondence: Jilu Shen, Tel +86 151 5515 2963, Email shenjilu@ahmu.edu.cn; 1464675852@qq.comIntroduction: As the last line of defense for clinical treatment, Carbapenem antibiotics are increasingly challenged by multi-drug resistant bacteria containing carbapenemases. The rapid spread of these multidrug-resistant bacteria is the greatest threat to severe global health problems.Methods: To solve the problem of rapid transmission of this multidrug-resistant bacteria, we have developed a rapid detection technology using CRPSPR-Cas12a gene editing based on multiple Recombinase polymerase amplification. This technical method can directly isolate the genes of carbapenemase-containing bacteria from samples, with a relatively short detection time of 30 minutes. The instrument used for the detection is relatively inexpensive. Only a water bath can complete the entire experiment of Recombinase polymerase amplification and trans cleavage. This reaction requires no lid during the entire process while reducing a large amount of aerosol pollution.Results: The detection sensitivity of this method is 1.5 CFU/mL, and the specificity is 100%.Discussion: This multi-scene detection method is suitable for screening populations in wild low-resource environments and large-scale indoor crowds. It can be widely used in hospital infection control and prevention and to provide theoretical insights for clinical diagnosis and treatment.

Aim: This study aimed to detect the carbapenem resistance of the Klebsiella pneumoniae strains, isolated from clinical specimens with genotypic and phenotypic methods. Material and Methods: A total of 87 Klebsiella pneumoniae strains whose carbapenem resistance was determined by disc diffusion method were included in the study. Carbapenemase was investigated using the combined disk method and polymerase chain reaction (PCR). Results: The evaluation of the PCR results demonstrated that OXA was detected in 60 (68.9%) samples, NDM was detected in 20 (22.9%), OXA + NDM in 5 (5.7%), and KPC was detected in 1 (1.1%) out of 87 clinical samples. Carbapenemase was not detected in one specimen with the PCR method. The results were found compatible with the combined disc test results for all isolates which were detected as only OXA, NDM, and KPC type carbapenemase positive. In 5 (5.7%) strains in which the co-existence of NDM and OXA type carbapenemases was detected by PCR, the combined disc method detected only OXA type carbapenemase. Conclusion: The combined disk method is inadequate in the presence of strains that have multiple carbapenemases, and also have OXA which is the most frequently detected carbapenemase in our hospital. EUCAST recommends verification by other methods in the presence of OXA-48. Genotypic methods can be used for confirmation testing. The detections of strains with NDM, multiple carbapenemases, and the first detection of KPC were striking in the study. Monitoring the spread of these strains in the hospital will be necessary for infection control.

Abstract This study aimed to explore the role of the two-component system Bae SR in the mechanism of drug resistance in carbapenem-resistant A. baumannii (CRAB) using molecular docking and real-time polymerase chain reaction (PCR). The two-component system Bae SR of Acinetobacter baumannii was subjected to molecular docking with imipenem, meropenem, and levofloxacin. Antibacterial assays and fluorescence quantitative PCR were used to explore protein–ligand interactions and molecular biological resistance mechanisms related to CRAB. The analysis of the two-component system in A. baumannii revealed that imipenem exhibited the highest docking energy in Bae S at − 5.81 kcal/mol, while the docking energy for meropenem was − 4.92 kcal/mol. For Bae R, imipenem had a maximum docking energy of − 4.28 kcal/mol, compared with − 4.60 kcal/mol for meropenem. The highest binding energies for Bae S–levofloxacin and Bae R–levofloxacin were − 3.60 and − 3.65 kcal/mol, respectively. All imipenem-resistant strains had minimum inhibitory concentration (MIC) values of 16 µg/mL, whereas levofloxacin-resistant strains had MIC values of 8 µg/mL. The time-sterilization curve showed a significant decrease in bacterial colony numbers at 2 h under the action of 8 µg/mL imipenem, indicating antibacterial effects. In contrast, levofloxacin did not exhibit any antibacterial activity. Fluorescence quantitative PCR results revealed significantly increased relative expression levels of bae S and bae R genes in the CRAB group, which were 2 and 1.5 times higher than those in the CSAB group, respectively, with statistically significant differences. Molecular docking in this study found that the combination of Bae SR and carbapenem antibiotics (imipenem, meropenem) exhibited stronger affinity and stability compared with levofloxacin. Moreover, the overexpression of the two-component system genes in carbapenem-resistant A. baumannii enhanced its resistance to carbapenem, providing theoretical and practical insights into carbapenem resistance in respiratory tract infections caused by A. baumannii.

Abstract Proteus mirabilis is a commensal bacterium dwelling in the gastrointestinal (GI) tract of humans and animals. Although New Delhi metallo-β-lactamase 1 (NDM-1) producing P. mirabilis is emerging as a threat, its epidemiology in our society remains largely unknown. LHPm1, the first P. mirabilis isolate harboring NDM-1, was detected from a companion dog that resides with a human owner. The whole-genome study revealed 20 different antimicrobial resistance (AMR) genes against various classes of antimicrobial agents, which corresponded to the MIC results. Genomic regions, including MDR genes, were identified with multiple variations and visualized in a comparative manner. In the whole-genome epidemiological analysis, multiple phylogroups were identified, revealing the genetic relationship of LHPm1 with other P. mirabilis strains carrying various AMR genes. These genetic findings offer comprehensive insights into NDM-1-producing P. mirabilis, underscoring the need for urgent control measures and surveillance programs using a “one health approach”.

Abstract Antimicrobial resistance genes (ARG), such as extended-spectrum β-lactamase (ESBL) and carbapenemase genes, are commonly carried on plasmids. Plasmids can transmit between bacteria, disseminate globally, and cause clinically important resistance. Therefore, targeting plasmids could reduce ARG prevalence, and restore the efficacy of existing antibiotics. Cobalt complexes possess diverse biological activities, including antimicrobial and anticancer properties. However, their effect on plasmid conjugation has not been explored yet. Here, we assessed the effect of four previously characterised bis(N-picolinamido)cobalt(II) complexes lacking antibacterial activity on plasmid conjugation in Escherichia coli and Klebsiella pneumoniae. Antimicrobial susceptibility testing of these cobalt complexes confirmed the lack of antibacterial activity in E. coli and K. pneumoniae. Liquid broth and solid agar conjugation assays were used to screen the activity of the complexes on four archetypical plasmids in E. coli J53. The cobalt complexes significantly reduced the conjugation of RP4, R6K, and R388 plasmids, but not pKM101, on solid agar in E. coli J53. Owing to their promising activity, the impact of cobalt complexes was tested on the conjugation of fluorescently tagged extended-spectrum β-lactamase encoding pCTgfp plasmid in E. coli and carbapenemase encoding pKpQILgfp plasmid in K. pneumoniae, using flow cytometry. The complexes significantly reduced the conjugation of pKpQILgfp in K. pneumoniae but had no impact on pCTgfp conjugation in E. coli. The cobalt complexes did not have plasmid-curing activity, suggesting that they target conjugation rather than plasmid stability. To our knowledge, this is the first study to report reduced conjugation of clinically relevant plasmids with cobalt complexes. These cobalt complexes are not cytotoxic towards mammalian cells and are not antibacterial, therefore they could be optimised and employed as inhibitors of plasmid conjugation.

Background: This study aimed to characterize carbapenem-nonsusceptible Acinetobacter (CNSA) isolated from patients with bacteremia from 1997 to 2015. Methods: A total of 173 CNSA (12.3%) was recovered from 1403 Acinetobacter isolates. The presence of selected β-lactamase genes in CNSA was determined by PCR amplification. The conjugation test was used to determine the transferability of metallo-β-lactamase (MBL)-carrying plasmids. Whole genome sequencing in combination with phenotypic assays was carried out to characterize MBL-plasmids. Results: In general, a trend of increasing numbers of CNSA was observed. Among the 173 CNSA, A. baumannii (54.9%) was the most common species, followed by A. nosocomialis (23.1%) and A. soli (12.1%). A total of 49 (28.3%) CNSA were extensively drug-resistant, and all were A. baumannii. The most common class D carbapenemase gene in 173 CNSA was blaOXA-24-like (32.4%), followed by ISAba1-blaOXA-51-like (20.8%), ISAba1-blaOXA-23 (20.2%), and IS1006/IS1008-blaOXA-58 (11.6%). MBL genes, blaVIM-11, blaIMP-1, and blaIMP-19 were detected in 9 (5.2%), 20 (11.6%), and 1 (0.6%) CNSA isolates, respectively. Transfer of MBL genes to AB218 and AN254 recipient cells was successful for 7 and 6 of the 30 MBL-plasmids, respectively. The seven AB218-derived transconjugants carrying MBL-plasmids produced less biofilm but showed higher virulence to larvae than recipient AB218. Conclusions: Our 19-year longitudinal study revealed a stable increase in CNSA during 2005–2015. blaOXA-24-like, ISAba1-blaOXA-51-like, and ISAba1-blaOXA-23 were the major determinants of Acinetobacter carbapenem resistance. MBL-carrying plasmids contribute not only to the carbapenem resistance but also to A. baumannii virulence.

Introduction: The dissemination of carbapenem-resistant Enterobacteriales (CRE) in nosocomial settings is primarily associated with the horizontal transfer of plasmids. However, limited research has focused on the in-host transferability of carbapenem resistance. In this study, ten isolates were collected from gut specimens of five individuals, each hosting two different species, including Escherichia coli, Klebsiella pneumoniae, Klebsiella aerogenes, Enterobacter cloacae, or Citrobacter koseri. Methods: Species identification and antimicrobial susceptibility were determined by MALDI-TOF MS and broth microdilution method. Carbapenemase genes were detected and localized using PCR, S1-PFGE and southern blot. The transferability of carbapenemase genes between species was investigated through filter mating experiments, and the genetic contexts of the plasmids were analyzed using whole genome sequencing. Results and discussion: Our results revealed that each of the ten isolates harbored a carbapenemase gene, including blaNDM-5, blaNDM-1, or blaKPC-2, on a plasmid. Five different plasmids were successfully transferred to recipient cells of E. coli, K. pneumoniae or A. baumannii by transconjugation. The genetic contexts of the carbapenemase gene were remarkably similar between the two CRE isolates from each individual. This study highlights the potential for interspecies plasmid transmission in human gut, emphasizing the colonization of CRE as a significant risk factor for the dissemination of carbapenemase genes within the host. These findings underscore the need for appropriate intestinal CRE screening and colonization prevention. [ABSTRACT FROM AUTHOR]

Objective: This study aims to conduct an in-depth genomic analysis of a carbapenem-resistant Proteus mirabilis strain to uncover the distribution and mechanisms of its resistance genes. Methods: The research primarily utilized whole-genome sequencing to analyze the genome of the Proteus mirabilis strain. Additionally, antibiotic susceptibility tests were conducted to evaluate the strain's sensitivity to various antibiotics, and related case information was collected to analyze the clinical distribution characteristics of the resistant strain. Results: Study on bacterial strain WF3430 from a tetanus and pneumonia patient reveals resistance to multiple antibiotics due to extensive use. Whole-genome sequencing exposes a 4,045,480 bp chromosome carrying 29 antibiotic resistance genes. Two multidrug-resistant (MDR) gene regions, resembling Tn6577 and Tn6589, were identified (MDR Region 1: 64.83 Kb, MDR Region 2: 85.64 Kbp). These regions, consist of integrative and conjugative elements (ICE) structures, highlight the intricate multidrug resistance in clinical settings. Conclusion: This study found that a CR-PMI strain exhibits a unique mechanism for acquiring antimicrobial resistance genes, such as blaNDM−1, located on the chromosome instead of plasmids. According to the results, there is increasing complexity in the mechanisms of horizontal transmission of resistance, necessitating a comprehensive understanding and implementation of targeted control measures in both hospital and community settings. [ABSTRACT FROM AUTHOR]